The in vitro and in vivo metabolic activation of the important antiviral agent ganciclovir is being examined. Currently, ganciclovir is under study as an antitumor agent for both animal and human tumors which have been transduced with the herpes simplex gene. The elevated levels of thymidine kinase encoded for by this gene can be used to enhance the phosphorylation of ganciclovir. This leads to a localized antitumor effect caused by the ability of ganciclovir triphosphate to inhibit tumor cell DNA polymerase and also to incorporate into tumor cell DNA. Since ganciclovir is initially phosphorylated by herpes thymidine kinase, its activation is slowed by agents which increase levels of dTTP (e.g. ribavirin), thus bringing about feedback inhibition of the enzyme. Tumor cells which have not been transduced but which are in close juxtaposition to HStk-transduced cells are also inhibited by ganciclovir (the "bystander effect"). Our studies indicate that the bystander effect is due to bulk transfer of ganciclovir mono-, di-, and triphosphates from transduced to non-transduced cells through gap junctions.